PROJECT SUMMARY/ABSTRACT Approximately 17,000 individuals each year are diagnosed with a malignant glioma in the United States and the vast majority of these patients will succumb to their disease. Given the lack of clinically available biomarkers for CNS malignancies, the conventional method for disease monitoring in these patients is radiographic. Unfortunately, anatomic changes detected by MRI and CT scans are often non-specific and lag behind progressing or regressing disease. Moreover, it can be difficult to discriminate between treatment effect and cancer growth with imaging alone. Patients must, therefore, have additional surgeries for definitive tissue diagnosis or inappropriately wait for radiographic findings to change as their disease progresses unabated. As a result, there is an incredible need for more sensitive and specific tumor biomarkers in neuro- oncology. We and others have shown that most cancers shed cell free molecules of tumor derived DNA into the circulation and that these molecules can be quantified as a measure of disease burden. Brain tumors are the exception to the rule and rarely shed detectable levels DNA into the bloodstream. However, we have provocative data to suggest that malignant gliomas shed cell free molecules of tumor derived DNA into the cerebrospinal fluid (CSF-tDNA). CSF-tDNA can be distinguished from DNA derived from normal cells by the presence of disease defining somatic mutations. Levels of CSF-tDNA can be quantified using sensitive digital sequencing based assays, such ?Safe-SeqS?. In Aim 1, we will use Safe-SeqS to quantify CSF-tDNA levels in longitudinal spinal fluid samples derived from 20 patients with malignant gliomas. We will determine how closely CSF-tDNA levels correlate with disease status as measured by clinical, radiographic and pathological findings. If successful, this approach will accurately assess tumor response and identify those patients at highest risk for recurrence, thus enabling the clinician to alter treatment regimens. There are also burgeoning data to suggest that all cancers, including malignant gliomas, evolve over time in response to various selection pressures, including treatment. These genetic changes have important clinical implications but currently there are no minimally invasive clinical assays that are capable of providing insights into the glioma genotype. As a result, in Aim 2, we will perform whole exome sequencing directly on the CSF and compare to exomic sequencing results from matched tumor/normal samples. This will allow us to understand what fraction of tumor genotype can be detected by analyzing the CSF directly. In order to determine the background mutation rate in CSF, in Aim 3 we will perform Safe-SeqS directly on the CSF of 50 individuals without any history of cancer. At the completion of this grant, we hope to validate CSF-tDNA as a candidate biomarker for individuals with malignant glioma and set the stage for large scale clinical trials that can be conducted to demonstrate clinical utility.